Ionizing radiation (IR) is a powerful medical treatment in the fight against cancer. The major cellular target of IR is DNA. Radiation induced DNA damage results in the activation of a complex network of intracellular processes that delay cell cycle progression, promote repair and if needed, execute the cell rather than allow genetically altered progeny to be formed. A major limitation to the use of radiation therapy is that it does not target significant physiological differences between normal and cancerous cells. Rational and effective improvements in the use of radiation therapy depend on understanding IR-mediated responses and on exploiting differences between the response of normal and cancerous cells. The p53 tumor suppressor, a major determinant of cellular responses to IR is not functional in the majority of human cancers, p53 controls IR induced pathways leading to cellular senescence and apoptosis. The absence of these pathways underlies the observation that tumors cells are often more resistant to the killing effects of IR than the normal tissue that surrounds them. However it also presents a significant physiological difference that can be exploited. In the absence of p53 the effect IR has on transformed cells is largely determined by the function of two human kinases, Chkl and Chk2 that are required for IR-induced cell cycle delays and repair. The I overall objective of this proposal is to understand how Chk1 and Chk2 protect normal and transformed cells from the genotoxic effects of IR. The project has three specific aims that address important unanswered questions concerning the function of Chkl and Chk2 in human cells. One aim is to determine how Chkl is regulated in response to IR, and to discover the role it plays in damage repair. The second aim is to determine the role Chk2 plays in maintaining genomic stability. The third aim is to identify novel substrates and regulators of the checkpoint kinases. Recent evidence that loss of Chk2 function radio-protects mice, without increasing the incidence of tumors, strongly suggests that the information generated by these studies is likely to aid in the rationale development of adjuvant agents that improve the efficacy of radiation therapy.